Evaluation and application of modularly assembled zincfinger nucleases in zebrafish

Cong Zhu, Tom Smith, Joseph Mcnulty, Amy L. Rayla, Abirami Lakshmanan, Arndt F. Siekmann, Matthew Buffardi, Xiangdong Meng, Jimann Shin, Arun Padmanabhan, Daniel Cifuentes, Antonio J. Giraldez, A. Thomas Look, Jonathan A. Epstein, Nathan D. Lawson, Scot A. Wolfe

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

Zinc-finger nucleases (ZFNs) allow targeted gene inactivation in a wide range of model organisms. However, construction of target-specific ZFNs is technically challenging. Here, we evaluate a straightforward modular assembly-based approach for ZFN construction and gene inactivation in zebrafish. From an archive of 27 different zinc-finger modules, we assembled more than 70 different zinc-finger cassettes and evaluated their specificity using a bacterial one-hybrid assay. In parallel, we constructed ZFNs from these cassettes and tested their ability to induce lesions in zebrafish embryos. We found that the majority of zinc-finger proteins assembled from these modules have favorable specificities and nearly one-third of modular ZFNs generated lesions at their targets in the zebrafish genome. To facilitate the application of ZFNs within the zebrafish community we constructed a public database of sites in the zebrafish genome that can be targeted using this archive. Importantly, we generated new germline mutations in eight different genes, confirming that this is a viable platform for heritable gene inactivation in vertebrates. Characterization of one of these mutants, gata2a, revealed an unexpected role for this transcription factor in vascular development. This work provides a resource to allow targeted germline gene inactivation in zebrafish and highlights the benefit of a definitive reverse genetic strategy to reveal gene function.

Original languageEnglish
Pages (from-to)4555-4564
Number of pages10
JournalDevelopment
Volume138
Issue number20
DOIs
StatePublished - Oct 15 2011

Keywords

  • Gata2
  • Vascular development
  • Zebrafish
  • Zinc-finger nuclease

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